Biology subjects

Klose, T.

Publications and source records attributed to Klose, T..

9 recordsLinked to original sources

bioRxiv2026

Agentic cryo-EM structure determination with CryoPilot

Single-particle cryo-electron microscopy (cryo EM) relies on mature computational tools, but experts must still inspect intermediate results, choose among alternative processing routes, and decide when earlier choices should be revised. Here we present CryoPilot, a system of two language model agents that carries cryo-EM data from raw micrographs through three-dimensional reconstruction to validated atomic models. Rather than executing a predefined pipeline, its reconstruction agent evaluates intermediate images and quantitative diagnostics, selects and parameterizes CryoSPARC jobs, tests alternative strategies, compares their outcomes, and retains, revises, or reverts decisions accordingly. A persistent programming environment and session database preserve the complete processing history, making each decision traceable and allowing multi day workflows to proceed with minimal human intervention. Its modeling agent determines map handedness, builds atomic models with CryoZeta, removes regions the density does not support, and optimizes each model by testing refinement steps against map fit, geometry, and DAQ, a measure of local model to map agreement. Across nine public EMPIAR datasets, eight reconstructions reached 2.61 to 3.51 A resolution, and refined models from seven maps agreed with reference structures to within 0.4 to 4.9 A Calpha; RMSD. For a challenging alpha latrocrustotoxin dataset, a single change of particle picking strategy that we suggested enabled CryoPilot to improve the reconstruction from 5.9 to 4.15 A. CryoPilot establishes an adaptive and auditable framework for agent-driven cryo-EM structure determination.

bioinformatics↗

bioRxiv2026

Analysis of the heterogenous structural states of the hexameric ATPase PilU of the Type IV pili from Vibrio cholerae

Type IV pili (T4P) mediate surface motility, host interactions, and DNA uptake through cycles of extension and retraction. While the primary retraction ATPase PilT has been extensively characterized, its homolog PilU remains less well understood despite being demonstrated as a PilT-dependent retraction ATPase. Here, we determined six PilU structures by cryo-electron microscopy and X-ray crystallography. The structures reveal a homohexameric assembly stabilized by interactions between the C-terminal catalytic domain of one subunit and the N-terminal PAS-like domain of a neighboring subunit. PilU adopts multiple conformational states, exhibiting digerent combinations of open and closed interfaces even in the absence of nucleotide. Comparison with PilT highlights structural features that likely underlie PilUs weak ATPase activity and its dependence on PilT for function. Together, these findings provide a structural framework for understanding PilUs role within the T4P retraction machinery.

molecular biology↗

bioRxiv2025

An unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis

Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy further reveals profound alterations in virion surface architecture. These findings uncover HxD as a previously unrecognized coatomer-targeting motif, highlighting an unexpected flexibility in coronavirus assembly pathways and broadening understanding of the cellular machinery that shapes coronavirus morphogenesis.

biochemistry↗

bioRxiv2023

Molecular basis for Gβγ-mediated activation ofphosphoinositide 3-kinase γ

The conversion of PIP2 to PIP3 by phosphoinositide 3-kinase {gamma} (PI3K{gamma}) is a critical step in neutrophil chemotaxis and is essential for metastasis in many types of cancer. PI3K{gamma} is activated via directed interaction with G{beta}{gamma} heterodimers released from cell-surface G protein-coupled receptors (GPCRs) responding to extracellular signals. To resolve how G{beta}{gamma} activates PI3K{gamma}, we determined cryo-EM reconstructions of PI3K{gamma}-G{beta}{gamma} complexes in the presence of various substrates/analogs, revealing two distinct G{beta}{gamma} binding sites, one on the p110{gamma} helical domain and one on the C-terminal domain of the p101 subunit. Comparison of these complexes with structures of PI3K{gamma} alone demonstrates conformational changes in the kinase domain upon G{beta}{gamma} binding similar to those induced by Ras{middle dot}GTP. Assays of variants perturbing the two G{beta}{gamma} binding sites and interdomain contacts that change upon G{beta}{gamma} binding suggest that G{beta}{gamma} not only recruits the enzyme to membranes but also allosterically controls activity via both sites. Studies in a zebrafish model examining neutrophil migration are consistent with these results. These findings set the stage for future detailed investigation of G{beta}{gamma}-mediated activation mechanisms in this enzyme family and will aid in developing drugs selective for PI3K{gamma}.

biophysics↗

bioRxiv2023

Isoform Specific Regulation of Adenylyl Cyclase 5 by Gβγ

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein Gs, but their response to G{beta}{gamma} regulation is isoform-specific. For example, AC5 is conditionally activated by G{beta}{gamma}. Here, we report cryo-EM structures of ligand-free AC5 in complex with G{beta}{gamma} and of a dimeric form of AC5 that could be involved in its regulation. G{beta}{gamma} binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the G{beta}{gamma} interaction with both purified proteins and cell-based assays. The interface with G{beta}{gamma} involves AC5 residues that are subject to gain-of-function mutations in humans with familial dyskinesia, indicating that the observed interaction is important for motor function. A molecular mechanism wherein G{beta}{gamma} either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core, is proposed. Because our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

biophysics↗

bioRxiv2021

Strain dependent structural effects and in vivo efficacy of enterovirus-D68 inhibitors

Acute flaccid myelitis (AFM) leads to loss of limb control in young children and is likely due to Enterovirus-D68 (EV-D68), for which there is no current treatment. We have developed a lead isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of the pleconaril-resistant CVB3-Woodruff (IC50 6-20 nM), EV-D68 (IC50 58 nM), and other enteroviruses. A mouse respiratory model of EV-D68 infection, in which pleconaril is inactive, showed decreased viremia of 3 log units as well as statistically significant 1 log reduction in lung titer reduction at day 5 after treatment with 11526092. A cryo-electron microscopy (cryo-EM) structure of EV-D68 in complex with 11526092 suggests that the increased potency may be due to additional hydrophobic interactions. Cryo-EM structures of 11526092 and pleconaril demonstrate destabilization of EV-D68 (MO strain) compared to the previously described stabilization of EV-D68 (Fermon strain) with pleconaril, illustrating clear strain dependent mechanisms of this molecule. 11526092 represents a more potent inhibitor in vitro with in vivo efficacy providing a potential future treatment for EV-D68 and AFM, suggesting an improvement over pleconaril for further optimization. One-Sentence Summary11526092 demonstrates protein destabilization, improved in vitro potency and in vivo efficacy when compared with pleconaril against EV-D68.

pharmacology and toxicology↗

bioRxiv2021

Structure of Vibrio phage XM1, a simple contractile DNA injection machine

Antibiotic resistance poses a growing risk to public health requiring new tools to combat pathogenic bacteria. Contractile injection systems, including bacteriophage tails, pyocins, and bacterial type VI secretion systems, can efficiently penetrate cell envelopes and become potential antibacterial agents. Bacteriophage XM1 is a dsDNA virus belonging to the Myoviridae family and infecting Vibrio bacteria. The XM1 virion, made of 18 different proteins, consists of an icosahedral head and a contractile tail, terminated with a baseplate. Here we report cryo-EM reconstructions of all components of the XM1 virion and describe atomic structures of 14 XM1 proteins. The XM1 baseplate is composed of a central hub surrounded by six wedge modules to which twelve spikes are attached. The XM1 tail contains a fewer number of smaller proteins compared with other reported phage baseplates, depicting the minimum requirements for building an effective cell-envelope-penetrating machine. We describe the tail sheath structure in the pre-infection post-infection states and its conformational changes during infection. In addition, we report, for the first time, the in situ structure of the phage neck region to near-atomic resolution. Based on these structures, we propose mechanisms of virus assembly and infection.

molecular biology↗

bioRxiv2020

A mysterious 80 nm amoeba virus with a near-complete "ORFan genome" challenges the classification of DNA viruses

Here we report the discovery of Yaravirus, a new lineage of amoebal virus with a puzzling origin and phylogeny. Yaravirus presents 80 nm-sized particles and a 44,924 bp dsDNA genome encoding for 74 predicted proteins. More than 90% (68) of Yaravirus predicted genes have never been described before, representing ORFans. Only six genes had distant homologs in public databases: an exonuclease/recombinase, a packaging-ATPase, a bifunctional DNA primase/polymerase and three hypothetical proteins. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a novel capsid protein with no significant homology with NCLDV major capsid proteins but with a predicted double-jelly roll domain. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists. Significance statementMost of the known viruses of amoeba have been seen to share many features that eventually prompted authors to classify them into common evolutionary groups. Here we describe Yaravirus, an entity that could represent either the first isolated virus of Acanthamoeba spp. out of the group of NCLDVs or, in alternative evolutive scenario, it is a distant and extremely reduced virus of this group. Contrary to what is observed in other isolated viruses of amoeba, Yaravirus is not represented by a large/giant particle and a complex genome, but at the same time carries an important number of previously undescribed genes, including one encoding a novel major capsid protein. Metagenomic approaches also testified for the rarity of Yaravirus in the environment.

microbiology↗

bioRxiv2018

Cryo-EM Structure of OSCA1.2 from Oryza sativa: Mechanical basis of hyperosmolality-gating in Plants

Sensing and responding to environmental water deficiency and osmotic stresses is essential for the growth, development and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis. Here, we report the cryo-EM structure and function of an ion channel from rice (Oryza sativa; OsOSCA1.2), showing how it mediates hyperosmolality sensing and ion permeability. The structure reveals a dimer; the molecular architecture of each subunit consists of eleven transmembrane helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The transmembrane domain is structurally related to the TMEM16 family of calcium dependent ion channels and scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that is parallel to the plasma membrane. These helical arms are well positioned to sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the transmembrane portion of the molecule to open the channel. Hydrogen-deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. The structure provides a framework to understand the structural basis of hyperosmolality sensing in an important crop plant, extends our knowledge of the anoctamin superfamily important for plants and fungi, and provides a structural mechanism for translating membrane stress to ion transport regulation.

biophysics↗